Fms-like tyrosine kinase receptor 3 (FLT3) is a Type III cell surface tyrosine kinase expressed on immature hematopoietic cells. The receptor is mutated in 25-30% of patients with acute myeloid leukemia (AML) by an internal tandem duplication (ITD) in the juxtamembrane region that perturbs a negative regulatory domain to enhance kinase activation.9,27,33 The mutant receptor is considered a model for tyrosine kinase activation and transformation in malignancy. The presence of mutated FLT3 in AML carries a poor prognosis and substantial work has been performed targeting this receptor with small molecule inhibitors. Most studies of FLT3 ITD propose that the receptor regulates cell growth or survival. However, we have recently had the opportunity to study patients treated with the second generation FLT3 inhibitor, AC220, also known as quizartinib. The overall clinical response to this compound in patients with relapsed FLT3 ITD mutant AML is greater than 70%. Interestingly, we have found that approximately half of patients respond with a cytotoxic response. In this scenario there is a rapid decrease in bone marrow blasts, transient myelosuppression and clearance of the FLT3 clone from blood and marrow. In contrast, other patients respond by differentiating their leukemic clone. Leukemic differentiation is characterized by a slow induction of differentiating forms in the marrow and persistence of FLT3 positivity. The difference between these patient groups is not associated with a difference in FLT3 allele burden or other clinical features. However, differentiating samples are more likely to have normal cytogenetics and carry mutations in DNMT3A. These data suggest that the combination of FLT3 ITD with DNMT3A expression alters the function of DNMT3A to make it primarily a regulator of myeloid differentiation rather than cell proliferation and survival. In order to pursu this hypothesis, we will study the effects of FLT3 ITD inhibition on patient samples on transcriptional regulators and epigenetic marks. We will also perform studies to recapitulate the growth promoting vs. differentiation regulating effects of FLT3 ITD by expressing FLT3 ITD alone or in combination with mutant DNMT3A in human CD34 stem/progenitor cells. Through these complementary approaches we will develop an enhanced understanding of FLT3 ITD regulation of myeloid signaling that will lead to improved therapies for patients with AML.